Application And Mechanism Of Laccase-mediator In The Removal Of Sulfonamides And Tetracyclines

Sulfonamide and tetracycline antibiotics are two kinds of broad-spectrum antibiotics,which are widely used in agriculture and animal husbandry to prevent and treat animal diseases.Since veterinary antibiotics are difficult to be metabolized and absorbed by animals,most of them are excreted into the environment in the form of parent compounds.The residual veterinary antibiotics in the environment could harm the ecosystem and human health.At present,the biological enzyme method,especially the oxidoreductases,is considered as a green tool to control the residues of veterinary antibiotics in the environment.In order to meet the needs of veterinary antibiotic residues in the large-scale industry,this research found a new laccase（Lyfu-Lac）from Lysinibacillus fusiformis.The catalytic properties of Lyfu-Lac were studied in detail.Furthermore,2,2-diazo-bis（3-ethyl-benzothiazole-6-sulfonic acid）diamine salt（ABTS）and syringic acid（SA）were selected as mediators to form two laccase-mediator systems with Lyfu-Lac.In this case,two kinds of common veterinary antibiotics（sulfonamides and tetracyclines）were successfully degraded.The degradation mechanism of sulfanilamide and tetracycline antibiotics by laccase-mediator system（LMS）was speculated through analyzing the change of parent compounds and products.The research contents of this topic are summarized as follows:Based on the NCBI database,the potential laccase gene（Gen Bank:AYW03784.1）from Lysinibacillus fusiformis was discovered through the homologous sequence search and analysis of probe enzyme.The potential laccase gene was connected to p ET-22b（+）and transformed into expression vector E.coli BL21（DE3）.The soluble expression of laccase was improved by optimizing IPTG concentration and induction temperature.Finally,Lyfu-Lac met the best soluble expression under the conditions of 0.5 mmol/L inducer concentration and 18℃induction temperature.After purification of the protein by Ni²⁺column affinity and SDS-PAGE detection,the results showed that the molecular weight of the single subunit of Lyfu-Lac protein was about 59 k Da.Based on the amino acid sequence alignment and phylogenetic tree analysis,the potential gene of Lyfu-Lac had a completely conserved HCH short peptide of laccase.According to the homologous modeling and dual-domain discovery,the active center of Lyfu-Lac contained three copper ion binding sites,T1,T2,and T3.It was speculated that the laccase was a new three-domain bacterial laccase.The catalytic properties of Lyfu-Lac were studied using ABTS as substrate.The results showed that the optimum p H and temperature of Lyfu-Lac were 4.0 and 80℃,respectively.Lyfu-Lac is strictly Cu²⁺dependent laccase.Lyfu-Lac could maintain stability for a long time at40℃,and maintain 71%residual enzyme activity over 12 h.Nano-DSC results showed that the T_m value of Lyfu-Lac was 88℃.The study of enzyme kinetics showed that the maximum reaction rate V_max,Michaelis-menten constant K_m,catalytic constant k_cat,and secondary rate constant k_cat/K_m of Lyfu-Lac were 0.045μmol·min^-1,0.0317±0.0011 mmol/L,249.10±0.04s^-1,7858.05±1.39 m M^-1·s^-1 respectively.ABTS and SA were selected as mediators to form laccase-mediator systems with Lyfu-Lac.Lac-ABTS system and Lac-SA system were used to degrade sulfonamides and tetracyclines antibiotics,respectively.The Lac-SA system composed of Lyfu-Lac and SA could degrade 87.5%sulfadiazine,92.2%sulfamethazine,and 86.5%sulfamethoxazole,respectively.Lac-ABTS,composed of Lyfu-Lac and ABTS,could degrade over 95%tetracycline and 83.1%oxytetracycline.The identification and analysis of the products by liquid chromatography-mass spectrometry found out the change of the substrate products during the degradation of sulfonamides and tetracycline antibiotics.It was speculated that the degradation process should be a two-step reaction:the first step is that Lyfu-Lac reacts with the mediator to produce radicals（dimethoxybenzoquinone,ABTS⁺·）;In the second step,free radicals react with the substrate to form the final product.In addition,there are two kinds of affinity in the reaction process:enzyme-mediater affinity（E-M,which determines the first reaction）and mediator-substrate affinity（M-S,which determines the second reaction）.